Device for detecting misfolded proteins and methods of use thereof

ABSTRACT

The present invention relates to diagnostic devices as well as methods of using these devices for detecting proteins of interest associated with diseases or disorders in mammals. In particular, the proteins of interest may be misfolded proteins associated with certain misfolded-protein disorders in mammals including those mammals suspected of or at risk of having such disorders.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/723,500, filed Dec. 20, 2020 which is adivisional of allowed U.S. patent application Ser. No. 15/211,957, filedJul. 15, 2016, which claims priority to U.S. Provisional ApplicationSerial No. 62/192,962, filed on Jul. 15, 2015, herein incorporated intheir entirety by reference.

BACKGROUND OF THE INVENTION

According to the American College of Obstetricians and Gynecologists,hypertensive disorders of pregnancy including preeclampsia complicateapproximately 10% of pregnancies throughout the world and are a leadingcause of maternal and fetal morbidity and mortality [ref: Hypertensionin Pregnancy, Report of the American College of Obstetricians andGynecologists' Task Force on Hypertension in Pregnancy, Obstetrics andGynecology 122 VOL. 122, NO. 5, Nov. 2013 (the ACOG 2013 guildelines)].Furthermore, these conditions are a leading cause of premature birthsand associated perinatal complications [ref: Ananth CV, Vintzileos AM. JMatern Fetal Neonatal Med. 2006:19(12):773-82]. Hypertension inpregnancy can be categorized as 1) preeclampsia-eclampsia, 2) chronichypertension 3) chronic hypertension with superimposed preeclampsia or4) gestational hypertension.

Preeclampsia-eclampsia is a poorly understood pregnancy-relatedcondition that is a leading cause of maternal mortality, prematurebirth, and rising healthcare costs for maternity. Globally, the death of76,000 expectant mothers is due to preeclampsia. Preeclampsia isresponsible for one-fifth of deaths related to pregnancy in the U.S.,and the condition can lead to seizures, organ failure and death. It mostcommonly occurs after about 20 weeks of pregnancy, and women are at riskthrough the postpartum period. The condition can result in seizures orconvulsions known as eclampsia. Preeclampsia may be categorized as mild,severe, less severe, more severe or as preeclampsia without severefeatures, or preeclampsia with severe features. HELLP syndrome, apreeclampsia subtype, is characterized as patients with symptoms ofhemolysis, elevated liver enzymes and low platelet count. Preeclampsiathat presents with an unusual compilation of symptoms is known asatypical preeclampsia.

The only known cure is to deliver the baby, and as a result,preeclampsia is the leading cause of pre-term births that are medicallyindicated, estimated to be 17% of all preterm births. Costs to the U.S.healthcare system are estimated to be over $13 billion for delivery andcare of mother and infant due to preeclampsia. Today, preeclampsiaremains a challenge to diagnose, as it is characterized only by itssymptoms: most often, high blood pressure and the presence of urineprotein. Research towards improving the diagnosis of preeclampsia hascommonly searched for known biomarkers in blood which are up- ordown-regulated, but few if any findings have yielded globally usefuldiagnostic products.

Research utilizing urine specimens of women with severe preeclampsiathat required medically indicated delivery due to a diagnosis ofpreeclampsia (MIDPE) and an unbiased mass spectrometry protein profilingapproach and found unique non-random cleavage products of SERPINA-1 andalbumin. Knowledge of the tendency of SERPINA-1 fragments to misfold andform supramolecular aggregates led to the proposal that preeclampsia maybe a misfolding disorder, not unlike Alzheimer's disease [See U.S. Pat.No. 8,263,342 and Buhimschi et al., Am J Obstet Gynecol. 2008 November;199(5): 551.e1-551.16. doi:10.1016/j.ajog.2008.07.006.]

Furthermore, misfolded protein based on binding of the proteins to CongoRed (CR) dye (“congophilia”) were found in urine from women withpreeclampsia. These misfolded protein(s) or “supramolecular aggregates”bound to conformational state-dependent anti-amyloid aggregateantibodies were associated with a highly active amyloid precursorprotein (APP) processing pathway and amyloid-like protein deposits inplacentas from preeclamptic women. [See Buhimschi et al., Sci. Transl.Med. 6, 245ra92 (2014).] A dot blot affinity assay measured theproportion of CR retained (due to binding to misfolded protein) afterwashing (as % of original CR) and results were reported as % Congo RedRetention (CRR).

In a feasibility study of 80 women (40 who required medically indicateddelivery and 40 were “control” healthy pregnancies), %CRR wassignificantly higher in severe preeclampsia urine (P<0.001) with 100%sensitivity and specificity. In a validation study of 582 women (incross sectional and longitudinal cohorts), women with severepreeclampsia and preeclampsia superimposed on existing high bloodpressure or proteinuria had higher %CRR than all other clinicalclassifications (P<0.001). Furthermore, 75% of women diagnosed with mildpreeclampsia, 89% with severe preeclampsia and 91% with superimposedpreeclampsia had CRR results higher than all other groups (P<0.05).Overall, CRR alone in the validation cohort had 85.9% sensitivity and85.00 specificity, positive likelihood ratio of 95% and negativelikelihood ratio of 95% in prediction of preeclampsia necessitatingMIDPE. CRR was superior to clinical screening methods currently used forpreeclampsia (P<0.001 compared to blood pressure or urine proteindipstick; P=0.004 compared to combined blood pressure combined withurine protein at American College of Obstetricians and Gynecologists(ACOG) recommended cutoffs) (Buhimschi et al.,Sci. Transl. Med. 6,245ra92 (2014) and U.S. Pat. No. 9,229,009.)

Congo Red also binds to cellulose which was used to create a simplepaper based assay. Normal urine-dye mixtures applied to test paperresults in dye binding to the cellulose in the paper visualized as a redtightly centered dot. In contrast, urine from women with congophilicurine proteins results in the dye no longer binding to cellulose becauseit is bound to the proteins and instead dispersing in a diffuse fashionvisualized as a halo. (See U.S. Patent Application Publication No.20150293115.) In a clinical study of 346 women referred to a labor anddelivery triage center to rule-out preeclampsia, patient urine wastested using the CR simple paper assay (CRD). The CRD test demonstrateda 79% sensitivity 89% specificity, negative predictive value of 91%,positive predictive value of 74% for the diagnosis of preeclampsia asdefined by the ACOG 2013 guidelines [ref: Rood et al 2016 AJOG Volume214, Issue 1, Supplement, Pages S24-S25]. The CRD test requires a stepof mixing urine with dye before applying the urine to the test paper andthe results can be challenging to read and interpret.

In view of the above, there is still a highly significant and unmet needfor a simple diagnostic device that may be used at the point of care todetect possible preeclampsia in pregnant mammals and especially women.Such a device could potentially save the lives of thousands of pregnantwomen as well as their unborn fetuses by providing early information asto whether a woman is at risk for preeclampsia or has preeclampsia andshould therefore receive immediate therapeutic intervention.

All patents and publications referred to herein are hereby incorporatedin their entirety by reference.

BRIEF SUMMARY OF THE INVENTION

The present invention includes a diagnostic device for detection of atleast one protein in a biological sample of a mammal. The devicecomprises a) a sample receiving material, wherein the sample receivingmaterial is capable of receiving a biological sample; b) a detectionreagent, which is reactive with (i.e., binds to) at least one proteinpresent in the biological sample; c) a trap which is in contact with thesample receiving material and is able to separate the detection reagentbound to the at least one protein in the biological sample from thedetection reagent that is not bound to the at least one protein in thebiological sample, whereby the detection reagent bound to the at leastone protein in the biological sample is able to flow through the trap,and whereby the detection reagent that is not bound to the at least oneprotein in the biological sample is captured by the trap; d) a capillarybed which is in contact with the trap, and is configured to contain thebiological sample after the biological sample flows through the trap.The capillary bed displays the bound detection reagent if the at leastone protein is detected in the biological sample. The sample receivingmaterial, trap, and capillary bed are configured to be in contact insequence. The sample receiving material of the device may comprise, forexample, the detection reagent. Also, the detection reagent may be on orwithin the sample receiving material.

Further, the device may be encased in a housing or cassette. The housingor cassette may comprise a well (or other entity) for biological sampleapplication and may also contain a window for reading the resultsobtained. The device may be used at the point of care in a variety ofclinical and non-clinical settings or in a clinical laboratory.

As noted above, the detection reagent binds to at least one protein inthe biological sample. This at least one protein may be, for example, amisfolded protein, a protein aggregate, a supramolecular proteinaggregate as well as mixtures thereof and fragments of each protein. Theat least one protein may comprise a beta sheet structure. Additionally,the at least one protein may be congophilic. The misfolded protein maycomprise, for example, alpha-1 antitrypsin (SerpinA1), ceruloplasmin,heavy-chain IgG, light-chain IgG, interferon-inducible protein 6-16(IF16-6, G1P3), albumin, mixtures thereof or fragments thereof, orfragments of each protein. However, the misfolded protein is not limitedto these proteins. For example, the misfolded protein may be any protein(or combination of proteins) which causes or is associated with aprotein-misfolding disorder.

The detection reagent may be, for example, an azo dye, Thioflavin T oran analog of an azo dye. An example of an azo dye that may be utilizedin connection with the present invention is Congo Red (i.e., di sodium4-amino-3-[4-[4(1-amino-4-sulfonato-naphthalen-2-yl)diazenylphenyl]phenyl]diazenyl-naphthalene-1-sulfonate).(An analog of Congo Red may also be utilized. For example, secondarydiazo dyes of the formula C₃₂H₂₂N₆Na₂0₆S₂ may also be used as thedetection reagent described herein in connection with the device of thepresent invention.) The Congo Red may be pre-loaded onto the samplereceiving material referred to above.

The sample receiving material of the device of the present invention maycomprise, for example, nitrocellulose, cellulose, a glass fiber, cotton,a woven mesh, a nonwoven material, a porous plastic, a polymer and/or apolyester. The polyester may be, for example, polyethylene.

The trap of the device of the present invention may comprise, forexample, nitrocellulose, cellulose, a glass fiber, a cotton/glass fiber,a woven mesh, a nonwoven material, a polymer, and/or a polysulfone.

The capillary bed of the device of the present invention may comprise amaterial such as, for example, nitrocellulose, a chromatographic paper,polysulfone and/or cellulose.

It should be noted that the device of the present invention provides atest result in approximately 10 minutes or less, preferablyapproximately 5 minutes or less, more preferably approximately 3 minutesor less, and most preferably 1 minute or less. Further, one may obtain aqualitative or semi-quantitative result by visualization. Moreover, onemay also obtain a semi-quantitative or quantitative result such that theamount of the at least one protein is measured, if desired.

The present invention also includes a diagnostic device, as describedabove, which is utilized for the detection of misfolded protein in abiological sample, for the detection of aggregated protein in abiological sample and/or for the detection of supramolecular aggregatedprotein in a biological sample, wherein the sample is obtained from amammal, for example, a human, primate or genetically-engineered mammal.In some instances, the mammal may be pregnant. The device, as describedabove, may be used for the detection of preeclampsia which may bediagnosed when the detection reagent is reactive (i.e., binds) to amisfolded protein, aggregate protein and/or supramolecular aggregateprotein (i.e., proteins associated with preeclampsia in pregnantmammals) contained with the biological sample. The trap of the devicemay be configured to competitively bind to the detection reagent of thedevice. The device may be configured as a lateral flow device or a stripcomprising the sample receiving material, the trap and the capillarybed.

Additionally the present invention encompasses a method of detecting atleast one protein in a biological sample of a mammal comprising thesteps of: a) applying a biological sample of said mammal to the samplereceiving material of the diagnostic device of the present invention fora time and under conditions sufficient to allow the at least one proteinto bind to the detection reagent; and b) detecting presence of detectionreagent on the capillary bed, wherein presence of detection reagent onthe capillary bed indicates presence of the at least one protein presentin said biological sample.

The method may be utilized for detecting at least one protein in abiological sample of a mammal having a protein-misfolding disorder or atrisk of having a protein-misfolding disorder. This method comprises thesteps of: (a) applying a biological sample of the mammal to the samplereceiving material of the diagnostic device described above for a timeand under conditions sufficient to allow the at least one protein tobind to the detection reagent and (b) detecting presence of bounddetection reagent on the capillary bed, wherein presence of detectionreagent on the capillary bed indicates presence of the at least oneprotein present in the biological sample, and indicates that the mammalhas the protein-misfolding disorder. Again, the at least one protein maybe, for example, a misfolded protein, an aggregated protein, asupramolecular aggregated protein, or a mixture thereof, or a proteinwith a beta sheet structure, such as a congophilic protein. The at leastone protein may be congophilic and/or may have a beta sheet structure.More specifically, the misfolded protein may be, for example, alpha-1antitrypsin (SerpinA1), ceruloplasmin, heavy-chain IgG, light-chain IgG,interferon-inducible protein 6-16 (IF16-6,G1P3), albumin, mixtures orfragments thereof, or fragments of each protein. The protein-misfoldingdisorder may be, for example, preeclampsia, Alzheimer's disease, priondisease or Parkinson's disease. Misfolded proteins found in otherdiseases or conditions characterized as protein-misfolding disorders mayalso be detected using the device of the present invention. As to thediagnosis of preeclampsia using the device of the present invention, onemay diagnose different forms of preeclampsia including, for example,mild preeclampsia, severe preeclampsia, atypical preeclampsia,hemodialysis-elevated liver enzyme-low platelet count (HELLP) syndromeand eclampsia. Further, a patient may be suffering from a hypertensivedisorder of pregnancy. Thus, the present method may be utilized todifferentially diagnose certain hypertensive disorders of pregnancy suchas differentiating preeclampsia from hypertensive conditions such aschronic hypertension or gestational hypertension or hypertension due toother causes, or differentiating the types of preeclampsia noted above.

The biological sample used in the above method and applied to the samplereceiving pad may be, for example, urine (clean or natural catch),blood, saliva, tissue, interstitial fluid, serum, plasma, cerebrospinalfluid, amniotic fluid or an extracted substance (e.g., extracted fromnasal secretions, ear wax, fecal material and tissue). The method isutilized in connection with biological samples from mammals, forexample, humans, primates and genetically-engineered mammals. The mammalmay be pregnant. In the case of a human, the method may be utilized inconnection with a pregnant woman who is approximately 8 to 42 weekspregnant (i.e., gestational age), preferably about 18 to 41 weekspregnant, and more preferably about 20 to 41 weeks pregnant or 20 weeksto delivery. However, the method of the present invention may also beutilized in connection with a postpartum mammal. It should be noted thatthe least one protein detected by the method, utilizing the device, maybe detected by visualization in order to obtain a qualitative orsemi-quantitative result or detected by measurement in order to obtain asemi-quantitative or a quantitative result. Subsequent to visualization,the at least one protein may be measured in order to obtain asemi-quantitative or a quantitative result.

Additionally, the present invention includes a kit comprising theabove-described device. This kit may also comprise a calibrator orcontrol reagent as well as instructions for use of the device. Also, thekit may comprise a sample applicator.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or patent application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIG. 1 shows photographs of device test strips of the invention in acassette or housing. The left panel of FIG. 1 illustrates the device ina housing prior to contact with a biological sample, with a sample well,a results window and control line. The right panel shows the deviceresults of testing urine samples known to be negative, weak positive andpositive for misfolded proteins associated with preeclampsia.

FIG. 2 shows an example of a test strip embodiment of the inventivedevice. The strip comprises a control dye, a display strip (or capillarybed), a trap (e.g., three pieces of material), a second sample pad (orsample receiving material), a first sample pad (or sample receivingmaterial) and the dye dried onto the sample pad.

FIG. 3 shows an embodiment of the diagnostic device of the presentinvention. FIG. 3A shows a top view with a sample pad, a trap and adisplay strip. FIG. 3B shows the same embodiment as in FIG. 3A viewedfrom the side. The sample pad, trap and display strip may be buttedtogether. FIG. 3C shows an embodiment with the sample pad, trap anddisplay strip overlapping each other (side view).

FIG. 4 shows different material configurations within the test strip.FIG. 4A shows an alternative embodiment of the invention having twosample pads, and a trap and display strip (capillary bed) in sequenceeach piece having an overlap with the adjacent piece. FIG. 4B showsanother, alternative embodiment of the invention having two sample pads,a triple trap and a display strip overlapping in sequence, with eachpiece having an overlap with the adjacent piece.

FIG. 5 shows an example of a dipstick configuration of the test strip ofthe present invention. This embodiment is configured with a cover tapeon top of the test strip. At the top of the test strip, the tape isopaque in color and may be used to hold the strip. In the middle, thetape is clear to provide a viewing window for the results. The tape maybe placed so that the clear window is, for example, approximately 10 mmabove the trap. The tape at the window may be surrounded by a color(e.g., white) which allows for easy viewing of the results. Below thetrap, the tape is opaque in color to cover the trap. At the bottom ofthe strip, the tape may display an arrow indicating which end of thestrip to dip into the test sample and may display a line indicating themaximum level to dip the test strip into the sample. The bottom end ofthe test strip may be free of cover tape to facilitate wicking of testsample upon dipping.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a device as well as methods of utilizing thisdevice. More specifically, the device is a lateral flow chromatographicrapid test that may be used in several clinical and non-clinicalsettings in order to detect proteins of interest in a biologicalspecimen. The device may be used to detect protein-misfolding disordersin mammals. The mammal may be suspected of having or at risk of havingone or more such disorders.

The device has several, different embodiments which will be describedherein. Basically, it comprises a test strip for detection of a proteinor proteins of interest in a biological sample (see, for example, FIG.1). The detection is carried out by means of a sequential series ofreactions. The test strip comprises a length of lateral flow assay orchromatographic material having capillarity and has a first end at whichchromatographic solvent transport begins. It also has a second end atwhich chromatographic solvent transport ends. The strip includes aplurality of zone or regions which are positioned between the first andsecond ends (see, for example, FIGS. 2 and 3). The zones include a firstzone which is impregnated with a detection reagent, for example, a dye.This detection reagent specifically binds with the protein or proteinsof interest in the biological sample. The first zone also receives thebiological sample. In comparison, the second zone, which is downstreamof the first zone, retains the detection reagent which is not bound tothe protein or proteins of interest in the biological sample whilepermitting detection reagent bound to the protein or proteins ofinterest in the biological sample to be transported to a third zone. Thethird zone of the test strip, located downstream of the second zone,receives the sample after it passes through the second zone. The thirdzone will display the detection reagent if the proteins of interest arepresent in the sample. It also comprises a means for detecting thedetection reagent bound protein as a measure of the protein or proteinsin the biological sample. In one embodiment, the device determines thepresence of misfolded proteins in the biological sample from a patientand allows for determination of whether a patient has or does not have aprotein-misfolding disorder. The presence of the protein or proteins canbe qualitatively or semi-quantitatively determined via visualization ormay be semi-quantified or quantified by use of a measuring entity whichmay be present within the device. After the biological sample is appliedto the first zone, the first zone releases the detection reagent in thesample, and the second zone separates the detection reagent bound to theprotein or proteins from unbound detection reagent, and permits onlybound detection reagent to be transported to the third zone which thendisplays the bound detection reagent for viewing or measurement. Thefirst zone may be a sample receiving material. The second zone may be atrap. The third zone may be a capillary bed or display strip.

The specific elements or components of the device and the characteristicor properties of these elements are described, in detail, as follows:

Materials Used In Components of Device

The sample receiving material, the trap, and the capillary bed can bemade from the same or different materials. The materials are generallyknown in the art of lateral flow devices and chromatography [see Ref:EMD Millipore Rapid Lateral Flow Test Strips Considerations for ProductDevelopment, available from EMD Millipore, Billerica, Mass.]. Membranesare selected based on physical and chemical properties that impactcapillary flow and therefore reagent deposition and assay performance.The materials include, for example, but are not limited to,nitrocellulose, filter papers, chromatography papers, cellulose, plasticpolymers, asymmetric polysulfone membrane, cotton, linters and/or glassfibers, polyesters, polyethylene and polysulfone. Membranes may be madeof polymers including, for example, nitrocellulose, polyvinylidenefluoride, nylon and polyethersulfone. Pad materials are often used assample receiving material to provide controlled and even receipt of thesample and facilitate flow to the contiguous strip materials of thedevice. The pad materials are porous, often made with cellulose (i.e.filter papers), glass fibers, woven meshes and synthetic nonwovenmaterial or polyesters. Filter matrices may be used for sample receiptparticularly if it is desirable to separate out extraneous materialcontained in the sample from that part of the sample to be assayed, forexample, to separate out cellular material from fluid. These filtermatrixes may be, for example, cellulose, asymmetric polysulfone membrane(including but not limited to Vivid™ Plasma separation membrane andasymmetric sub-micron (BTS) polysulfone membrane). Absorbent pads may beused, for example, as a wick at the end of the device strip to pullsample through the lateral flow strip, and may increase the amount ofsample assayed and enhance assay sensitivity. These absorbent pads areoften cellulose or cotton linters and optimally selected based onthickness, compressibility, and uniformity of bed volume. The entirestrip may be assembled on a backing card often a card of a plasticbacking and adhesive. While these various materials are often thought offor specific purposes in lateral flow devices, as described herein, eachmaterial may be considered for suitable properties for the purposes ofthe receiving material, trap and display strip of the present invention.See Examples for further discussion of materials.

Other materials utilized in the configuration of the test strip,specifically the display strip, are known in the art of lateral flowtechnology and chromatography.

Elements or Components of the Device Sample Receiving Material (SamplePad)

The first element of the device (hereinafter referred to as the samplereceiving material or the sample pad) acts as a sponge and holds anexcess of sample fluid to be tested. The receiving material absorbssample, but also permits it to flow or to wick to the next contiguousmaterial. It is typically inert to, and thus does not react with,proteins of interest that may be present in the sample, as well as thedetection reagent (e.g., dye), allowing proteins and detection reagentto flow or to wick through the material to contiguous material in thelateral flow device.

The sample receiving material may be dipped into the sample from themammal or patient or, alternatively, the sample may be indirectly ordirectly applied to the sample receiving material. The sample may beapplied to the sample receiving material by, for example, a dropper witha metered tip, a pipette, a transfer pipette, or a pipette capable ofrepeated dispensing of the patient sample. If the sample receivingmaterial is configured to be dipped into the biological sample, (seeFIG. 5, for example), then the sample receiving material may berelatively long (for example, but not limited to, about 10 mm). If thesample receiving material is configured to receive the sample, appliedby, for example, a dropper or pipette, then the sample receivingmaterial may be relatively short (between, but not limited to, about 5mm and about 10 mm). Commonly, the width may be from 2 mm to 10 mm, andmost commonly, 2.5 to 5 mm (±0.5 mm). Variations in the length and widthof the sample receiving material are possible and depend upon suchfactors as the size of the cassette or housing as well as the ability ofthe biological sample to sufficiently mix with the detection reagent.

In particular, the sample receiving pad acts as a capillary matrix inwhich the biological sample and a detection reagent (e.g., dye) canfreely mix. The sample pad may also have a detection reagent in a driedformat suitable for an optimized chemical reaction between the analyte(e.g., protein of interest to be detected in the biological sample) andthe detection reagent. The detection reagent may be pre-loaded onto thesample receiving material. In one embodiment, the biological sample(e.g., the urine) when added to the sample receiving pad dissolves thedetection reagent, and then the sample and detection reagent dye mix aretransported across the device by flowing through the sample pad tocontiguous material such as the trap.

Alternatively, the sample pad comprises a series of two or more samplepads (see, for example, FIG. 4). For example, the first pad may receivethe sample and the second may contain the detection reagent, whereby thebiological sample migrates from the first to the second element (pad)containing a detection reagent in a dried format suitable for anoptimized chemical reaction between the analyte of interest and thedetection reagent. If two or more sample receiving materials areutilized, either one may comprise the detection reagent. Preferably, thefirst sample receiving material comprises the detection reagent and thesecond sample receiving material does not.

In yet another embodiment, a first sample pad receives the biologicalsample and is designed to separate out or retain insoluble material thatmay be present in the sample. The filtered sample then flows through thefirst sample pad or to the second pad and the detection reagent isincorporated in either the first or second sample pad and allows forsuitable mixing of the detection reagent and sample. The second pad mayhave the same or different composition as the first pad.

Further, in yet another embodiment, the first sample pad receives thesample and also contains the detection reagent, and the second padprovides for additional time for the mixing of detection reagent withthe analyte of interest before entering the next contiguous material inthe strip for example, the trap.

In an additional embodiment, the sample receiving material comprises asubstrate for the detection reagent and retains the substrate upondrying. The detection reagent may be on or within the sample receivingmaterial. Once the patient sample is added, the detection reagent isreleased. The substrate does not react with or absorb the patient samplewhich, when applied, moves through the matrix and onto contiguousmaterial, for example, the trap.

It should be noted that the sample may be applied or placed into acassette or housing, for example, through a sample well or other entityof the device for receiving the sample. The sample well or entity may bepositioned over the sample receiving material of, for example, a teststrip when it is assembled or encased inside a cassette or housing. (SeeFIG. 1.)

Materials useful as sample receiving material or sample pads aregenerally known in the art of lateral flow devices and chromatography[see Ref: EMD Millipore Rapid Lateral Flow Test Strips Considerationsfor Product Development, available from EMD Millipore, Billerica, Mass.]and are selected based on physical and chemical properties that impactsample receipt, controlled and even capillary flow, and samplefiltering. Additionally, if the sample receiving material also containsthe detection reagent, ideally, the material is a suitable matrix forholding the detection reagent and optimally releasing it upon additionof the test sample. The pad materials are porous, often made withcellulose (e.g., filter papers), glass fibers, woven meshes, synthetic,nonwoven material or porous plastic, for example, polyesters. Othermaterials that can be used as sample receiving material are, forexample, polysulfone asymmetric membranes, cotton/glass fibers materialssuch as Ahlstrom® 8950, plastic polymer membranes, for example,polyethylene, (e.g. high density polyethylene), polytetrafluoroethylene,and porous glass fiber membranes (see, for example Porex, Fairburn,Ga.). See Examples for further description of materials (i.e., Examples2 and 6).

It should be noted that the sample receiving material used in a deviceof the present invention, when the detection reagent is a dye withaffinity to cellulose, may be cellulose provided enough detectionreagent is present for binding to the modified protein or proteins ofinterest in the biological sample. More specifically, the cellulosecannot be permitted to “out compete” the detection reagent in connectionwith binding to the protein of interest, e.g., the misfolded protein orproteins in the biological sample. Alternatively, cellulose may be usedfor the sample receiving material if it is present in a matrix which isless reactive with the detection reagent than the modified proteins orprotein of interest. (Cellulose, for purposes herein, is defined as anorganic compound with the formula (C₆H₁₀O₅)n and, in particular, is apolysaccharide consisting of a linear chain of several hundred to manythousands of β(1->4) linked D-glucose units.)

Trap

Next, the fluid (e.g. sample or sample mixed with detection reagent)flows from the sample receiving material or sample pad through a filter(hereinafter referred to as a “trap”) designed to retain any unbounddetection reagent. In particular, the trap serves to separate freedetection reagent (e.g., dye) from protein-bound detection reagent inthe lateral flow device. Specifically, the trap material permits theflow of sample through to the next contiguous material but retains,retards the flow of, or binds to the unbound detection reagent if theprotein or proteins of interest are not present in the biologicalsample.

The trap abuts but preferably overlaps with the sample pad that containsthe detection reagent (e.g., dye) or the series of sample pad elements(see, for example, FIG. 3). The trap functions to separate the detectionreagent that is bound to the test sample protein or proteins of interest(e.g., misfolded protein or proteins) from detection reagent that is notbound to test sample protein or proteins, thus permitting the bounddetection reagent to flow through while retaining the unbound detectionreagent. Alternatively, the trap may be a series of one or more filtersof the same or different materials.

Not to be bound by theory, the trap material may contain a substrate forthe detection reagent such that unbound detection reagent binds the trapmaterial and does not flow to the next material. Detection reagent thatis already bound to proteins does not bind to the substrate in the trapand does flow to the next material in the strip. The substrate may bethe trap material (e.g. cellulose) or it may be a chemical modificationor addition to the trap material. Alternatively, a structural feature ofthe trap material composition may provide for the retention of unbounddetection reagent.

Also, the trap may be comprised of multiple pieces of materialoverlapped or in succession to optimize for retaining unbound detectionreagent (see FIG. 4). The multiple pieces may be the same or made fromdifferent materials. Filter matrices may be used for the trap, forexample cellulose, thermoplastic polymers such as asymmetric polysulfonemembrane (including but not limited to Vivid' plasma separation membraneand asymmetric sub-micron (BTS) polysulfone membrane). See Examples forfurther description of trap materials (e.g. Examples 2, 4, 5, 8, 13, 14and 16).

It was unexpected and quite surprising that there were, indeed, manynitrocellulose materials that actually worked well and permitted flow ofurine and dye-bound proteins through the device. For example, Whatman®AE99 nitrocellulose membrane worked very well. (See Table 1.) There werealso cellulose materials that worked reasonably well (for example,Ahlstrom® 601, 319, 247, Whatman® CF1, CF3, CF4; EMI11513, 5475, 5493),but there were also some cellulose materials that did not perform well(for example, Ahlstrom® 270). Surprisingly, among the materials thatperformed well for allowing protein-bound CR dye to flow while retainingunbound dye were Vivid™ Plasma separation materials (Pall Corporation)and asymmetric sub-micron (BTS) polysulfone membrane (Pall Corporation).(See Tables 1 and 2.)

When the detection reagent is a dye such as Congo red, the trap materialmay be, for example, filter papers, cellulose based and/or materialssuch as EMI 11513, EMI 5475, EMI 5493, 1281, 642, Standard 17, C048,LF1, LF1, VF2, CF1, CF3, Ahlstrom 319. The trap may be, for example,about 5-10 mm in length, or it may be a series of pieces each 5-10 mm inlength.

In one embodiment, the trap retains free detection reagent (e.g., dye)but allows protein-bound dye to flow through. In a specific embodiment,the trap is comprised of cellulose and the detection reagent is CongoRed.

Detection Reagent

The detection reagent is a substance which is reactive with a protein orproteins of interest in the sample. For example, the detection reagentmay be a substance which is reactive with or has a binding affinity forthe misfolded protein or proteins (e.g., congophilic proteins),aggregated proteins and/or supramolecular aggregated proteins present ina biological sample from a mammal, e.g. the patient sample. Thedetection reagent may be preloaded onto a reagent pad (for example,applied onto the reagent pad, or the reagent pad dipped into thedetection reagent or dye. The reagent pad may be the sample receivingmaterial or sample receiving pad.

In one aspect, the detection reagent is a dye that stains the subset ofproteins of interest in the biological sample, if present. In oneembodiment, the detection reagent may react with misfolded proteins. Forexample, the dye may be an azo dye such as Congo Red (CR), or analogthereof, either buffered or unbuffered. Alternatively, other dyes couldbe used as detection reagents as long as these dyes have an affinity for(and can bind to or react with) the misfolded proteins, aggregatedproteins and/or supramolecular protein or proteins of interest in thebiological sample or patient sample. Examples of such dyes include butare not limited to Congo Red analogs such as those described in thefollowing publications: Sellarajah S et al, Synthesis of analogues ofCongo red and evaluation of their anti-prion activity, J Med Chem. 2004Oct. 21; 47(22):5515-34; and Helene Rudyk et al, Screening Congo Red andits analogues for their ability to prevent the formation of PrP-res inscrapie-infected cells, Journal of General Virology (2000), 81,1155-1164. The detection reagent for detecting misfolded protein orproteins may also be, for example, Thioflavin T.

Further, in one aspect of the invention, the detection reagent ispresent in a dried form in the device, but may be present in other formsas well. The form of detection reagent is suitable for optimally mixingwith the sample when applied and allowing binding to the protein ofinterest. The form of the detection reagent is suitable for long termstability or shelf life of the device. In one embodiment, the drieddetection reagent is a dye. Furthermore, the dye may be an Congo Red andmay be present in the device in an amount of, for example, 0.1 ug to 800ug, more preferably, 0.2 ug to 480 ug, even more preferably lug to 400ug, and even more preferred 2.5 to 120 ug.

Congo Red (CR) (e.g., buffered or non-buffered) may be pre-applied tothe sample receiving material. For example, a CR solution can be appliedto the material during kit manufacture and dried before assembly andpackaging (see FIG. 2 and Example 6).

The detection reagent is detectable, i.e., visible to the naked eye, orotherwise detected, for example, by visual examination and/or mechanicalor electronic reader(s).

The present invention provides for the detection reagent to beincorporated into the test device, for example, during manufacturing orassembly of the device. This is an improvement over previous devices fordetecting misfolded proteins, where dye and biological sample needed tobe mixed prior to adding to a test. In a preferred embodiment, the teststrip contains a sample receiving material that contains the detectionreagent. When the biological sample is added to the test device, intothe sample receiving material, it mixes with the detection reagent inthe sample receiving material and the biological sample-detectionreagent mixture flows through the device.

Capillary Bed (“Display Strip”)

A sample (e.g., urine) with or without protein-bound-detection reagentpasses through the trap and onto a capillary bed (hereinafter referredto as a “display strip”) where it accumulates. Thus, the display strippermits the flow of sample up the strip and displays the presence orabsence of the detection reagent. In particular, the display strippermits the flow of sample up the strip and displays the presence orabsence of the detection reagent -bound analyte. The detection reagentor bound reagent can then be visualized by human or mechanical means (toobtain a qualitative result) and/or then measured (i.e.,semi-quantitatively or quantitatively). The display strip optimallyprovides for even flow of the sample throughout and relativelyhomogeneous display of the detection reagent when protein of interest ispresent. In certain embodiments, the intensity or concentration of thedetection reagent on the display strip may correspond to the amount ofprotein of interest in the sample. In another embodiment, the distancethe detection reagent flows up the display strip may be indicative ofthe amount of protein of interest in the biological sample. Furthermore,both the intensity of detection reagent and the distance up the displaystrip may be indicative the concentration of proteins in the sample.Furthermore, in the case of detection of misfolded protein aggregates orsupramolecular aggregates, both the intensity of detection reagent andthe distance up the display strip may be indicative the size of proteinaggregates present in the sample. Suitable materials for the capillarybed include, for example, nitrocellulose or chromatography papers. Also,polysulfone asymmetric membranes provide suitable display strips. Othersuitable materials include CytoSep® membranes such as CytoSep® 1660 andMN-260. The capillary bed of the lateral flow strip is aligned under aresults viewing window of the lateral flow strip cassette or housing,described below. See Examples for further details of materials (e.g.Example 2, 3 and 9). If protein bound detection reagent is present, thenthe detection reagent is visualized within the window. (See FIG. 1.)

Wick

Optionally, the device contains a wick. The wick may be positioned afterthe third zone or the display strip in the strip or device of theinvention. When in use, the biological sample (e.g., fluid) applied tothe device continues to migrate from the display strip into a finalporous absorbent material, the “wick”, that acts as a sample accumulatorand also may function to pull sample along the strip. Absorbent pads maybe used, for example, as a wick at the end of the device strip to pullsample through the lateral flow strip, and may increase the amount ofsample assayed and enhance assay sensitivity. These absorbent pads areoften cellulose or cotton linters and optimally selected based onthickness, compressibility and uniformity of bed volume.

Backing Card

The device may have a backing card. The entire strip may be assembled ona backing card (for example, those available from Lohmann, Orange, Va.)which is often a card of a plastic backing and adhesive.

Housing/Cassette

In one embodiment of the present invention, the diagnostic device ishoused, encased or encapsulated in a housing or cassette. The device mayfurther comprise a housing or cassette, including but not limited to, acartridge, plastic device or extruded plastic piece configured for thepurpose, that encases the device. Several generic cassette housings arecommercially available (for example, from Kanani Biologicals, Gujarat,India or EASE-Medtrend Biotech LTD, Shanghai, China) or may becustom-produced for the purpose at hand. (See, for example, U.S. DesignPatent Appln. Ser. No. 29/533,647.) The device may be configured in ahousing or cassette having a sample well for receiving the sample,wherein the sample well is positioned over the sample receiving materialof the strip when it is assembled/housed inside the cassette.Furthermore, the device may be configured such that, when assembled inthe housing or cassette, the display strip or capillary bed of the stripis positioned beneath a result viewing window of the housing.

In one embodiment, the device of the invention also comprises anelectronic reader that is able to quantify the result, e.g. theintensity of the detection reagent (e.g., dye) on the capillary bed(i.e., in the results window) and may further comprise a display screen(e.g., an LED screen) to display the results. This reader may be part ofthe housing, or an element that is assembled integrally with thehousing, for example, in the results display window. Such readers aredescribed, for example, in Venkatraman, Biosensors and BioelectronicsVolume 74, 15 Dec. 2015, Pages 150-155, PCT Application No. W02013083686A1, PCT Application No. WO2004010143 A2, and PCT Application No.WO2006010072 A2.

Cover Tape

The device with or without a housing may further comprise a cover suchas a protective adhesive tape (for example, available from Lohmann,Orange, Va.) or other material capable of protecting the device fromdamage and providing for proper reading of test results. For example,the device may be configured to be used as a dipstick such as a urinedipstick. In this embodiment, the lateral flow strip may be covered witha protective adhesive tape. (See FIG. 5 and Example 11)

Run Control Reagent

In a preferred embodiment, a control reagent may be present on thecapillary bed visible in the view window before the device is used. (SeeFIG. 1 and FIG. 2, Example 12.) When the biological sample (e.g., fluid)flows through the capillary bed, the control reagent dissolves, thecontrol reagent line disseminates and/or the control is carried away, upthe display strip so that no control reagent is visible in the resultswindow, or it is blurred, or some other difference may be visualized ormeasured. The change in the control reagent in the view window indicatesthat the test sample (e.g., fluid) was added and has run through thedevice properly, e.g., serves as a run control. (See Example 12.) Thecontrol reagent is detectable visually e.g. by the naked eye orotherwise detected or measured, for example, by mechanical examinationand/or an electronic reader. In one embodiment, the control reagent istartrazine. Other dyes that may be used as the run control reagentinclude: FD&C Blue No. 1—Brilliant Blue FCF, E133 (blue shade), FD&CBlue No. 2—Indigotine, E132 (indigo shade), FD&C Green No. 3—Fast GreenFCF, E143 (turquoise shade), FD&C Red No. 3—Erythrosine, E127 (pinkshade), FD&C Red No. 40—Allura Red AC, E129 (red shade), FD&C Yellow No.5—Tartrazine, E102 (yellow shade), FD&C Yellow No. 6—Sunset Yellow FCF,E110 (orange shade).

Detected Proteins of Interest

As noted above, the present invention is directed to a device andmethods of utilizing this device for detection of proteins of interest,more specifically, misfolded proteins, aggregated proteins and/orsupramolecular protein aggregates. It is known that the alpha helix isthe prominent structural motif of the functional protein in its nativeconformation. In contrast, a conformational change in a protein can leadto a beta sheet structural motif (i.e., beta sheet structure) or amisfolded protein that then tends to cause protein aggregation andtoxicity. The misfolded proteins may therefore be in the form of proteinaggregates or supramolecular aggregates and may be associated withmisfolded protein disorders such as preeclampsia, Alzheimer's disease,prion disease and Parkinson's disease.

In particular, these misfolded proteins, protein aggregates and/orsupramolecular aggregates associated with preeclampsia which aredetected by the device and methods of the present invention may include,but are not limited to, for example, alpha-1 antitrypsin (SerpinA1),ceruloplasmin, heavy-chain IgG, light-chain IgG, interferon-inducibleprotein 6-16 (IF16-6,G1P3), albumin as well as fragments of eachprotein, mixtures thereof, and fragments of such mixtures. Theseproteins have binding affinity for the detection reagent (to bedescribed below) utilized in the device of the present invention. Forexample, these misfolded proteins are congophilic, having an affinityfor the dye referred to as Congo Red.

The device of the present invention may also be used to detectprotein-misfolding disorders other than preeclampsia. For example, thedevice may be utilized to detect misfolded proteins in such misfoldedprotein disorders or conditions as Alzheimer's disease, Cerebralbeta-amyloid angiopathy, Retinal ganglion cell degeneration in glaucoma,Prion diseases, Parkinson's disease and other synucleinopathies,Tauopathies, Frontotemporal lobar degeneration (FTLD), FLTD-FUS,Amyotrophic lateral sclerosis (ALS), Huntington's disease and othertriplet, repeat disorders, Dementia (familial British and Danish),Hereditary cerebral hemorrhage with amyloidosis, CADASIL, Alexanderdisease, various amyloidoses, Serinopathies, Type II diabetes, Inclusionbody myositis/myopathy, cataracts, Retinitis pigmentosa with rhodopsinmutations, Medullary thyroid carcinoma, Pituitary prolactinoma,Hereditary lattice corneal dystrophy, Mallory bodies, Pulmonary alveolarproteinosis, Odontogenic tumor amyloid, Cystic fibrosis, Sickle celldisease and Critical illness myopathy.

Biological Sample

The protein or proteins of interest to be detected may be found in abiological sample from a mammal. The biological sample may be, forexample, urine obtained from a clean or natural catch, cerebrospinalfluid, amniotic fluid or any bodily fluid sample potentially comprisingthe protein or proteins of interest (e.g., blood, saliva, amnioticfluid, cerebrospinal fluid, plasma or serum). The sample may also be anextract of excretions from a patient, for example, from nasalsecretions, fecal material, or ear wax, or tissue specimens extractedwith appropriate solutions and applied to the device. The proteins ofinterest may be found, for example, in a biological sample from apregnant or postpartum mammal.

Patient

The patient may be a mammal. Furthermore, the mammal may be pregnant,for example, a pregnant woman, a pregnant primate or agenetically-engineered animal model designed to have the physicalsymptoms and signs of preeclampsia such as those utilized in laboratorystudies (e.g., high blood pressure and protein in the urine).Preferably, for the diagnosis of preeclampsia, the patient may be anypregnant woman. The pregnant woman may be suspected of havingpreeclampsia or at risk of having preeclampsia. For example thesuspicion may be based upon the following: 1) exhibiting the signs andsymptoms of preeclampsia, for example, as set forth in the AmericanCollege of Obstetrics and Gynecology Guidelines (ACOG) (Hypertension inPregnancy, Report of the American College of Obstetricians andGynecologists' Task Force on Hypertension in Pregnancy, Obstetrics andGynecology 122 VOL. 122, NO. 5, NOVEMBER 2013), specifically, forexample TABLE E-1 (the “ACOG 2013 guidelines”) and/or 2) having one ormore risk factors for preeclampsia (e.g., a woman having a previouspregnancy involving preeclampsia, a woman carrying multiple fetuses, awoman with cardiovascular or renal abnormalities or a woman having anautoimmune disease such as lupus).

Kit

The present invention also includes a kit for detecting proteins ofinterest in a sample. In one embodiment, the kit comprises a device fordetection of misfolded proteins associated with preeclampsia, present ina sample from a pregnant mammal. The kit comprises the device of theinvention as described above in any alternative embodiments describedabove. The kits may also comprise a means for applying the patientsample to the sample receiving material, for example, a pipette (forexample Fine tip transfer pipette available from Genesee Scientific, SanDiego, Calif.) or dropper, a control as well as instructions for use ofthe device. Thus, not only may the device be utilized as a stand-aloneentity, it may also be used in kit form which may be more advantageousin some clinical or non-clinical settings. The kit may be packaged in afoil or mylar pouch. Kit pouches may furthermore be packaged singly, orin multiples, e.g., 2, 5, 10, 15, 25, 50 or 100 kits per package.

Settings For Use of Device and Methods Utilizing Device

The device of the present invention may be used in, for example,clinical laboratories (either within a hospital setting or outside ahospital setting), immediate care settings, physician officelaboratories, emergency departments (e.g., within a hospital) or as anear-patient testing or point-of-care device by medical personnel,non-medical professionals or even by the patient herself when thepatient is a human. Further, the device may be used in combination withother diagnostic assays (e.g., immunoassays such as those that detectother proteins (i.e., biomarkers) associated with preeclampsia such as,for example, sFlt-1, P1GF, PP-A, PP13, pentraxin, inhibin-A and solubleendoglin), and/or other methods or observations commonly utilized in thediagnosis of preeclampsia (e.g., blood pressure readings, clinical testsused in the diagnosis of preeclampsia including platelet count, serumcreatinine concertation, serum ALT (alaninine aminotransferase) and AST(aspartate aminotransferase), and other signs or symptoms such as weightgain, dizziness, headaches, blurred vision, etc.

For example, the present invention includes a method of diagnosis ofpreeclampsia or performing a differential diagnosis in a patientsuffering from a hypertensive disorder of pregnancy or a patient who maybe suspected of having preeclampsia comprising the steps of: a)determining a blood pressure of the pregnant patient, wherein a bloodpressure greater than 140/90 mm/Hg in the pregnant patient may indicatepreeclampsia in the pregnant patient and b) applying a biological samplefrom the patient to the device of the present invention, wherebydetection of at least one protein of interest in the biological sampleof the patient provides or supports a diagnosis of preeclampsia in thepatient or acts as a differential diagnosis.

Further, the present invention also encompasses a method of treating apregnant mammal suspected of having preeclampsia comprising the stepsof: a) applying a biological sample from the mammal to the device of thepresent invention in order determine the presence of the at least oneprotein indicating the pregnant mammal has preeclampsia; and b)delivering the pregnant mammal in order to treat the preeclampsia.Further, the device may also be used post-delivery to determine if thepatient is expressing the misfolded protein or proteins in thebiological sample. If the misfolded protein or proteins are present,further patient management or therapeutic intervention may be needed(e.g., administration of magnesium sulfate or other anti-hypertensiveagents) to treat the preeclampsia or the patient may be monitored usingthe device of the present invention to indicate when proteins are nolonger detectable in the biological sample. Additionally, the device ofthe present invention may be utilized after therapeutic intervention todetermine if treatment was successful or to measure a change (e.g.,decrease, absence or increase) in the amount or presence of protein ofinterest present in the biological sample. Thus, the device of thepresent invention may be used pre- and post-treatment of the patient todetermine whether further therapeutic intervention is necessary, todetermine whether therapeutic intervention has been effective and/orwhether to administer an alternative form of therapeutic intervention oran increased dosage of the therapeutic agent to resolve the preeclampsiaor other protein-misfolding disorder.

Advantages of Device and Methods Utilizing The Device

The devices of the invention are advantageous over existing devices forat least the following reasons: 1) the device provides for a simplifiedtesting procedure with fewer steps; 2) the device provides for the useof standardized test materials for optimal manufacturing; 3) the deviceproides for improved stability of the detection reagent in the test kitto provide a longer shelf life; 4) results are simpler and easier toread all while 5) retaining relatively low cost; and 6) providing fastresults suitable for point-of-care use or use in clinical laboratorysettings.

In particular, the provided device and methods are vastly superior toknown paper kits for detecting possible preeclampsia (see, e.g., U.S.Patent Appin. Publication No. 20150293115) in that the provided devicerequires fewer steps for the user, results are easier to read and ismore stable than the paper kits resulting in a long shelf life of atleast 6 months, preferably 1 year, even more preferably 2 years, evenmore preferably 3 years, even more preferably 4 years, and even morepreferably 5 years. The device of the invention also provides fastresults (i.e., within 3 minutes, preferably within 2 minutes, and morepreferably within 1 minute or less from application of the biologicalsample (e.g., urine) to the device) suitable for point-of-care userswith minimal training.

The present invention may be illustrated by the use of the followingnon-limiting examples:

EXAMPLES Example 1 Sample Devices

FIG. 1 shows photographs of device test strips of the invention in acassette or housing. The left panel of FIG. 1 illustrates the device ina housing prior to contact with a biological sample. The right panelshows the device results of testing urine samples known to be negative,weak positive and positive for misfolded proteins associated withpreeclampsia.

FIG. 2 shows an example of a test strip embodiment of the inventivedevice. The strip comprises a control dye, a display strip, a trap(e.g., three pieces of material), a second sample pad, a first samplepad and the dye dried onto the sample pad.

FIG. 3A shows one embodiment of the diagnostic device of the presentinvention. FIG. 3A shows a top view. Figure B shows the same embodimentas in FIG. 3A but viewed from the side. The sample pad, trap and displaystrip may be butted together. FIG. 3C shows an embodiment with thesample, pad, trap and display strip overlapping each other (side view).

FIG. 4 shows different material configurations within the test strip.FIG. 4A shows an alternative embodiment of the invention having twosample pads, a trap and a display strip (capillary bed) in sequence.Each piece has an overlap with the adjacent piece. FIG. 4B showsanother, alternative embodiment of the invention having two sample pads,a triple trap and a display strip which overlap in sequence, each piecehaving an overlap with the adjacent piece.

FIG. 5 shows an example of a dipstick configuration of the test strip ofthe present invention. This embodiment is configured with a cover tapeon top of the test strip. At the top of the test strip, the tape isopaque in color and may be used to hold the strip. In the middle, thetape is clear to provide a viewing window for the results. The tape maybe placed so that the clear window is, for example, approximately 10 mmabove the trap. The tape at the window may be surrounded by a color(e.g., white) which allows for easy viewing of the results. Below thetrap, the tape is opaque in color to cover the trap. At the bottom ofthe strip, the tape may display an arrow indication as to which end ofthe strip to dip into the test sample and may display a line indicatingthe maximum level to dip the test strip into the sample. The bottom endof the test strip may be free of cover tape to facilitate wicking oftest sample upon dipping.

Example 2 Materials Testing

Various paper-like materials were tested for ability to differentiateurine samples from pregnant women with and without preeclampsia. CongoRed (CR) dye (Sigma. St. Louis, MO) was added to urine samples and adrop was added to the material to assess characteristics suitable forsample receiving, trap and display strip. The resulting spot wasvisualized after about 3 minutes and evaluated for a visual differencebetween urine from preeclampsia patients and that from normal controlpregnancies (Table 1). A result of “excellent” indicates the materialwas suitable in providing a visually observable difference betweenCR-urine from preeclampsia patients and CR-urine from control pregnantpatients such that the material could be useful in the diagnostic testdevice. A result of “poor” indicates the material was not suitable inproviding a visually observable difference between CR-preeclampsiapositive and CR-control urines. From a scale from a result of “poor”being the least suitable material, “subtle” was slightly better but notideal, “okay” better yet, “good” even better, “better” more improved and“excellent” being the most suitable materials. It was determined thatthe polysufone asymmetric materials provided the best results. Certaincotton materials such as CF3 and CF4 worked well whereas other cottonslike Ahlstrom 270 did not work well. Nitrocellulose and glass fibermaterials generally did now work well for this purpose.

TABLE 1 Manufacturer Material Composition Results Pall Corp, Port Vividpolysulfone asymmetric Excellent Washington, New York Pall Corp BTSpolysulfone asymmetric Excellent Whatman, GE AE98 Nitrocellulose OkayHealthcare Bio- Sciences, Pittsburgh, PA Whatman AE99 NitrocelluloseBetter Whatman FF120 nitrocellulose on plastic Good Whatman FF170HPnitrocellulose on plastic Poor Whatman FF85 nitrocellulose on plasticSubtle Whatman FF80HP nitrocellulose on plastic Subtle Whatman Prima 40Nitrocellulose Okay Whatman Prima 125 Nitrocellulose Less so WhatmanImmunopore RP Subtle Whatman Standard 14 glass fiber Poor WhatmanStandard 17 glass fiber Poor Whatman CF3 Cotton Okay Whatman CF4 CottonOkay Whatman VF2 Poor Whatman LF1 Subtle Millipore, EMD HF75Nitrocellulose Modest Millipore, Billerica, MA Millipore HF90Nitrocellulose Modest Millipore HF120 Nitrocellulose Poor MilliporeHF135 Nitrocellulose Poor Millipore HF170 Nitrocellulose Poor MilliporeHF180 Nitrocellulose Poor Sartorius Stedim, CN150 Nitrocellulose SubtleBohemia, New York Sartorius Stedim, CN140 Nitrocellulose SubtleSartorius Stedim, CN95 Nitrocellulose Subtle Ahlstrom, 270 Cotton PoorHelsinki Finland Ahlstrom  222 Poor Ahlstrom  320 Poor Ahlstrom  111Glass Poor Ahlstrom  142 Glass Poor Ahlstrom  21 Poor Ahlstrom  141Glass Poor Ahlstrom 6613 glass or polyester Poor Ahlstrom 6615 PoorAhlstrom  181 Poor Ahlstrom  169 Poor Ahlstrom  161 Poor Ahlstrom  151Poor Ahlstrom  131 Poor Ahlstrom  601 Cotton Good Ahlstrom  237 OkayAhlstrom  238 Cotton Subtle Ahlstrom 8950 cotton or glass Poor Ahlstrom8951 glass fiber or polyester Poor Ahlstrom 8964 glass fiber PoorAhlstrom CytoSep 1662 Proprietary Good Ahlstrom CytoSep 1663 ProprietaryGood Ahlstrom CytoSep 1660 Proprietary Good Ahlstrom ReliaFlow 319Subtle Ahlstrom ReliaFlow 800 Poor Ahlstrom ReliaFlow 1281 SubtleMacherey Nagel MN-260 Good Bethlehem, PA Macherey Nagel MN-321 SubtleMacherey Nagel MN-615 Good Macherey Nagel MN-616g Okay Macherey NagelMN-640 Okay Macherey Nagel MN-6176 Okay Lypore 9334 Poor Rochester, NHLypore 9390 Poor Lypore 9389 Poor

Example 3 Test Strip

Test strips were assembled using a Ahlstrom® 8950 (a cotton/glass fibercomposition)(“8950”) sample pad and a BTS or Vivid™ running strip orcapillary bed, as BTS and Vivid had demonstrated excellent results inproviding a visually observable difference between CR-urine frompreeclampsia patients and CR-urine from control pregnant patients. CongoRed was added to urine samples, the sample was vortexed, and thensamples were applied to the 8950 end of the test strip. Results wereobserved on the BTS or Vivid™ membrane run strip. Urine from women withpreeclampsia applied to Vivid™ membrane containing strips resulted invery clear signal (pink/red Congo Red staining) with negative urinesshowing no signal. Signal was volume dependent with decreasing volumesresulting in decreasing signal. The BTS strips showed no differencebetween positive and negative urines with both resulting in positivestaining at the higher sample volume. Signal decreased equally for thepositive and negative urines with decreasing sample volumes applied.

TABLE 2 Positive Staining Negative Run Material Urine Results UrineResults Vivid 90 ul Positive 90 ul Negative Vivid 50 ul Weak positive 50ul Negative Vivid 30 ul Negative 30 ul Negative BTS 90 ul Positive 90 ulPositive BTS 50 ul Weak positive 50 ul Weak positive BTS 30 ul Negative30 ul NegativeThese results indicate that the Vivid™ material was suitable in thediagnostic test device of the present invention whereas the BTS did notperform well.

Example 4 Trap Materials Evaluation

Vivid™ and BTS membranes were evaluated as materials for the trap, thatis positioned between the sample pad and the run strip (i.e., capillarybed or display strip) to determine if they aided in the retention of CRdye when negative urines (i.e., urine not containing the proteins ofinterest from a woman without preeclampsia) were applied, allowing CRwith preeclampsia-positive urine to flow through to the display strip.Test strips were assembled to include 8950 as a sample pad with eitherVivid™ membrane or BTS membrane followed by a CytoSep® 1660 run strip.Urine with added CR was applied to the 8950 and results were observed onthe CytoSep® 1660 run strip.

CR staining was seen on all strips when positive urine (i.e., urinecontaining the proteins of interest from a woman withpreeclampsia)—Congo Red dye was applied. While strips made with a Vivid™membrane trap were negative when negative urine-Congo Red was applied,strips made with a BST membrane trap showed some dye staining. TheVivid™ material was an effective trap to retain CR dye when negativeurine was tested, while letting through the CR dye when positive urinewas tested. BTS was a less effective trap.

Example 5 Evaluation of Materials for use for Pre-Loaded DetectionReagent in Test Strips

Congo Red dye was applied to sample pad materials and allowed to dry.Positive urine was applied and the results on the dye release from thesample pad were observed.

TABLE 3 Material Results POREX ® 4894 (Porex, Good dye release and flowFairburn, GA) Porex ® X-4897 Good dye release and flow Porex ® D3883BModest dye release, spot still retained Porex ® PVA Poor dye release, noflow Fusion 5-1 (GE Healthcare Modest dye release spot Life Sciences,Pittsburgh still retained PA) Fusion 5-2 (GE Healthcare Modest dyerelease spot Life Sciences,) still retained GF DVA 1 (GE Healthcare Poordye release Life Sciences,) GF DVA 2 (GE Healthcare Poor dye releaseLife Sciences,) Whatman ® 33 (GE Modest dye release, spot HealthcareLife Sciences,) still retained

The detection reagent (i.e., dye in this instance) ideally is releasedfrom the sample pad material upon application of the biological sample,i.e., urine. Several materials including POREX® 4894 and Porex® X-4897provided for good dye release and therefore are highly suitable in thediagnostic test device. Additionally, several materials also provideddye release and are suitable, such as Fusion materials (glassmicrofibers) and Whatman® 33.

Example 6

Evaluation of Sample Pads with Dye Incorporated Into Test Strips

Sample pad materials including Porex®, X4897 and POREX® 4894 infusedwith Congo Red dye were incorporated into test strips with a Vivid™ xtrap and an MN-260 run or display strip. Urine was added to the samplepad of each strip and results observed on the MN-260 display strip.

Results: The sample pad materials released dye when urine was added.Adding preeclampsia-positive urines resulted in positive staining of theMN260 material. In most cases, adding preeclampsia-negative urinesresulted in no staining of the MN260; however, in some cases there wassome staining at the beginning of the MN260 strip. These resultsindicate that the configuration of Porex®, Vivid™, MN260 materials couldprovide means for differentiating preeclampsia positive frompreeclampsia negative urines, providing for a useful diagnostic testdevice.

Example 7 Test Strip Evaluation in Lateral Flow Assay Cassette

Strips were assembled of dye infused POR-4899 sample pad, placed on topof Fusion 5, followed by Vivid X membrane serving as a trap and thenMN260 display strip. Strips were placed in generic cassette with thesample pad directly underneath sample port of the cassette and the MN260visible in the cassette results window. [75 ul] Seventy-five microlitersof urine were added in the sample port and results were observed in theresults window. When positive urines were added. Red staining wasvisible in the results window whereas, when negative urines were added,no staining was observed. This configuration provided for an effectivediagnostic device differentiating preeclampsia positive frompreeclampsia negative urines.

Example 8 Refining the Trap

At times, negative urine tested on the strip resulted in some streakingof dye up the results strip, particularly over time. Additional trapconfigurations were evaluated to see if better separation of positivevs. negative samples could be achieved and if this streaking withnegative urines could be eliminated. Strips were assembled with dyeinfused POR-4899 sample pad placed on top of Fusion 5, a trap of twooffset but overlapping layers of Vivid™ X and MN260 results strip. Whentested by applying negative urines, the double layer trap prevented dyeleakage and streaking thereby providing for an improved diagnostic testdevice for differentiating preeclampsia positive from preeclampsianegative urines.

Example 9 Device Strip Configurations

POR-4899 was striped with Congo Red dye solution dispensed at 6 μl/cmusing a Kinematic dispensing machine. POR-4588 was striped at 4 μl/cm.Pads were dried at 37° C. for 1 hour and stored in a desiccant cabinet.Test strips were assembled using the striped POR material, one layerVivid™ X trap and MN260. The POR-4899 strip performed well when testingurines; however, the strip containing POR-4588 resulted in dye leakageonto the display strip when testing negative urines. POR-4588 wasfurther evaluated when striped at 4 μl/cm and resulted in clean negativeurine run. Then, POR-4588 was evaluated when striped at 6 or 8 μl/cmassembled with a double Vivid™ trap. This strip assembly with 6 μl/cmdye strip gave the best results when testing positive urines and theleast amount of dye leakage or streaking when testing negative urines.Further, evaluation of strip with a 3 layer Vivid™ trap and POR-4588striped at 8 μl/cm gave good results and a stronger positive signal.

TABLE 4 Congo red Pos/neg Dye pad dispensing rate Trap differentiationPOR-4899 6 ul/cm Single Vivid ™ trap √ POR-4588 4 ul/cm Single Vivid ™trap False positive POR-4588 6 ul/cm Double Vivid ™ √ trap POR-4588 8ul/cm Double Vivid  ™ √ trap

The device configured with POR-4599 with Congo Red applied at 8 ul/cmand a double vivid trap resulted in a clean, negative result whenpreeclampsia negative urine was tested and a strong positive result whenpreeclampsia positive urine was tested and therefore is a preferreddiagnostic test device configuration.

Example 10 Evaluation of a Dipstick Format Test Design

Test strips were assembled using POR-4588 striped at 6, 8 or 10 μl/cmwith a 3 layer Vivid™ trap and MN260. Strips were dipped into 60 ul ofurine. Urine successfully wicked up the strip and the version with 8μl/cm stripping provided the best positive test results with cleannegative test results. Therefore, a suitable dipstick format of thediagnostic test device can be configured in this way and is useful fordifferentiating preeclampsia positive urine from preeclampsia negativeurine for diagnostic purposes.

Example 11 Further Evaluation of Diagnostic Test Device Configuration

Test strips made of two Porex® sample pads, one striped with dye; 3Vivid™ layer trap, and MN260 were tested in a cassette/housing and asdipsticks for testing urine. The cassette housing improved results,perhaps because of controlled flow of sample onto central spot on samplepad, good uptake of dye, flow up center of strip, and more homogeneousstaining of run strip result. Five negative urines all tested negative;5 positive urines resulted in 2 weak positive, 3 positive results. As adipstick, the test strip was placed into 100 μl urine in a test tube. At3 minutes, all results for both positive and negative urines werenegative. However, given more time, by 5 minutes the positive urinesresults were 2 weak positives, 3 positives, while negative urinesremained negative. Therefore, these test configurations are suitablepreeclampsia diagnostic test devices.

Example 12 Evaluation of Run Control

The purpose of this experiment was to investigate a control thatindicates when the sample has run up the strip successfully, a “runcontrol”. When a negative biological sample is tested and no staining isobserved on the display or results strip it is difficult to tell if itis a negative result, or the test failed to run correctly (i.e., nosample was actually added, insufficient sample was added or insufficientwicking of the sample up the strip occurred). A stripe of a watersoluble coloring was sprayed across the run strip material, positionedso that it would be visible in the top of the cassette results windowwhen assembled in the housing. Urine samples were applied to the samplepad through the sample port and flow observed through the resultswindow. When sample reached the control dye stripe, the stripe dissolvedand ran with the sample. The disappearance of the run control stripeprovided an easily visible indication that sample had run through thestrip successfully. The control did not interfere with reading resultsof the urine test. An example of control strip dye is tartrazine. Otherdyes that may be used as the run control include: FD&C Blue No.1—Brilliant Blue FCF, E133 (blue shade), FD&C Blue No. 2—Indigotine,E132 (indigo shade), FD&C Green No. 3—Fast Green FCF, E143 (turquoiseshade), FD&C Red No. 3—Erythrosine, E127 (pink shade), FD&C Red No.40—Allura Red AC, E129 (red shade), FD&C Yellow No. 5—Tartrazine, E102(yellow shade), FD&C Yellow No. 6—Sunset Yellow FCF, E110 (orangeshade).

Example 13 Investigation of Alternative Materials for Trap

Test strips were assembled with 2 Porex® sample pads, the first stripedwith Congo Red (CR) dye, a variety of alternative trap materials aslisted in Table 5 and MN260 run strip. Negative urine sample were testedon the assembled test strips and the ability of each trap to: 1) retainthe CR dye when proteins of interest in the sample are not present and2) not retain protein bound CR were evaluated.

TABLE 5 Results testing Results testing Trap Material Negative samplePositive sample EMI 11513 (EMI Specialty Negative, slight dye PositivePapers, Redding, CT) run off EMI 5475 (EMI Specialty Negative, verylittle Positive Papers) dye run off EMI 5493 (EMI Specialty Negative,slight dye Positive Papers) run off ReliaFlow 1281 Negative, slight dyePositive (streaky) run off Ahlstrom 642 Negative, very little Positivedye run off Whatman ® Standard 17 Negative, modest Positive (streaky)dye run off Millipore C048 Negative, very little Weak positive dye runoff Whatman ® LF1 Negative, slight dye Weak positive run off Whatman ®VF2 Negative, very little Weak positive dye run off Whatman ® CF1Negative, slight dye Positive run off Whatman ® CF3 Negative, verylittle Positive dye run off

Results: Most of these trap materials showed promise in differentiatingcongophilic protein-positive biological samples (congophilic proteinpositive urine) from congophilic protein-negative biological samples(congophilic protein-negative urine).

To further improve the retention of detection reagent (CR) when negativesamples are tested, test strips were then made using select trapmaterial described in Table 6 in three layers.

TABLE 6 Results testing Negative Results testing Positive Trap Materialsample sample EMI 11513 Negative Weak Positive 642 Negative PositiveC048 Negative Weak positive CF1 Negative, slight run off Positive

Results: A triple layer trap was effective in retaining unbound CRdetection reagent from entering the display strip. However, triple trapconfiguration made with EMI 11513 and C048 also reduced signal resultsintensity when testing positive samples. Triple traps made with 642provided the best results for both positive and negative samples,amongst those traps tested.

Example 14 Further Investigation of Trap Materials

To further investigate suitable materials for use as a trap in thediagnostic test device of the present invention, strips with 2 Porex®sample pads, the first striped with CR dye (8 ul/cm), a trap asdescribed in Table 7 and MN260 run strip were assembled. Strips of eachconfiguration were tested with a congophilic negative urine sample, twoweak positive congophilic protein samples and two strong positivecongophilic protein samples.

TABLE 7 Investigation of trap materials as single layer Strong StrongMaterial Neg Weak positive Positive Weak positive Positive EMI 11513 −+− +− ++ 237 − + ++ + ++ 319 − + ++ + ++ CF1 − +− ++ +− + Vivid ™ − ++++ +− ++ 5475 − + ++ +− ++ 5493 − +− ++ +− ++ C048 − +−− + +−− + CF3 −+−− + +−− + 238 − + + +− + 601 − + + + + 1281 − +− + +− ++ 642 − +− ++−− + MN640m − +− + +−− + MN615 − +− ++ +− +

Surprisingly, 5493 results were less intense than 5475, with 5493 about4 times more absorbent and 2 times faster capillary rise characteristicsthan 5475. These results suggest that less absorption and a slowercapillary rise is preferable for robust signal. Similarly, CF3 is weakerthan CF1, having two times the absorption. Material 238 has a weakersignal than 237 and has twice the wick rate, and 319 showed thestrongest color results of this paper line and had the slowest wickrate. MN640 which is a faster running material has weaker signal thanMN615.

Results show that the materials that have less absorbency, slowercapillary rise or wick rate may be preferred materials for more robustsignal.

Example 15 Evaluation of Diagnostic Test Devices

A complete test strip was assembled, comprising a dye infused Porex®sample pad placed on top of Fusion 5 (a proprietary single layer matrixmembrane made by GE), a Vivid™ membrane strip trap and MN260 capillarybed membrane. The strip was housed in a generic LFA cassette case. Urinesamples were added to the circular sample well and results were observedin the rectangular window at 3 minutes.

Results: No dye was observed in the window for negative urine, a lightpink was observed for weak positive urine and a strong pink was observedfor the strong positive urine sample. These results indicate this is asuitable configuration for a diagnostic test device for detection ofcongophilic proteins in a sample for the diagnosis of preeclampsia.

Example 16 Further Evaluation of Optimal Trap Materials

A complete test strip was assembled, comprising a dye infused Porex®sample pad a second Porex® pad (without dye), a trap of single or doublelayers of 11513, 5475, 319, 247, or CF1, a MN260 membrane capillary bedwith a wick of Whatman 470. The MN260 contained a run control stripe oftartrazine dye. The test strip was housed in a LFA-like cassetteoptimized for addition of test sample onto dye infused Porex® pad, and aview window centered over the MN260 membrane such that the tartrazinedye is observable prior to running the test and not observed after thetest is run. Urine samples were added to the circular sample well andresults were observed in the rectangular window at 3 minutes.

Results

No dye was observed in the window for negative urine, a light pink wasobserved for weak positive urine and a strong pink/red was observed forthe strong positive urine sample at 3 minutes. Furthermore, the yellowtartrazine dye stripe was evident prior to adding sample, could beobserved to travel along with the wicking of the sample, and was barelyvisible or completely gone at 3 minutes. These results indicate this isa suitable configuration for a diagnostic test device for detection ofcongophilic proteins in a sample for the diagnosis of preeclampsia.

Example 17 Results of Present Device Versus CRD Test Results

The test device of the present invention was evaluated by testing 105clinical urine specimens from a study of the Congo Red dot (CRD) test inwomen suspected of preeclampsia (Rood et al., 2016 AJOG, 214(1)s24-s25).Samples were chosen to provide 27 strong positives, 28 negatives and 50weak positive samples. The negative/positive status of the samples wasdetermined by %CRR (Irina A. Buhimschi et al., Sci. Transl. Med. 6,245ra92 (2014)). Samples were added to the test device and results werevisually scored at 3minutes as either negative (− or 0), weak positive(+ or 1) or strong positive (++ or 2).

Results showed that the test device results were concordant with the CRDresult in 103/105 of the samples (98%). The test result of the device ofthe present invention, when compared to an adjudicated diagnosis ofpreeclampsia (based on ACOG guidance: Hypertension in Pregnancy Reportof the American College of Obstetricians and Gynecologists' Task Forceon Hypertension in Pregnancy, 2013 Obstetrics & Gynecology 122(5):1122-1131), showed a sensitivity of 91%; specificity of 64%, accuracy of82% with a positive predictive value of 83% and a negative predictivevalue of 79%.

Upon adding the test sample to the sample well of the test, the front ofthe liquid traveling up the strip was visible by 30 seconds. The fronthit the yellow dye control stripe at about 1 minute and traveled to theend of the window by about 1.5 min. Strong positive results were clearlyvisible with the front of liquid traveling into the results window by 30seconds and beyond. The full results window was colored by 1.5 min. Weakpositive results take longer to resolve than the strong positives as theliquid front commonly appeared negative with color developing in theresults window at about 1-3 minutes after addition of the sample. FIG. 1shows examples of results.

Example 18 Evaluation of Device Using Urine Sample Controls

The device test strip was assembled, comprising a Porex® 4588 sample paddye infused 120 ug/device, a second Porex® pad (without dye), a trap ofdouble layers of Ahlstrom 319, a MN260 membrane capillary bed with awick of Whatman 470. The MN260 contained a run control stripe oftartrazine dye. The test strip was housed in a LFA-like cassetteoptimized for addition of test sample onto dye infused Porex® pad, and aview window centered over the MN260 membrane such that the tartrazinedye is observable prior to running the test and not observed after thetest is run.

Urine specimens from five (5) patients with confirmed preeclampsia werepooled to create a positive assay control. The presence of congophilicproteins was confirmed by the Congo Red dot blot test (Buhimschi 2014).Phosphate buffered saline (10 mM phosphate, pH 7.4, 150 mM NaCl) wasused as a negative control and for making the dilution series of thepositive assay control. The following dilution were prepared (in ratioof sample to total volume of buffer): undiluted, 1:4, 1:8, 1:15, 1:40.Each urine sample of the dilution series was tested in duplicate on thedevice.

Results: Each replicate for each urine sample for a particular dilutionexhibit the same density of staining on the devices. The results of thetesting are presented in the following table.

TABLE 8 Testing results from a dilution series of a pooled urine sampleDilution Test Results Undiluted +++ 1:4 ++ 1:8 + 1:10 ± 1:15 ± 1:40 −

These data demonstrate that the diagnostic device can detect congophilicproteins, and that the effect is titratable. These data also indicatethat device exhibits a progressive visual scale of red colorcorresponding to increasing concentration of congophilic proteins.

What is claimed is:
 1. A method of detecting of at least one protein ina biological sample of a mammal, comprising: a) applying a biologicalsample of said mammal to said sample receiving material of saiddiagnostic device of claim 1 for a time and under conditions sufficientto allow said at least one protein to bind to said detection reagent;and b) detecting presence of detection reagent on the capillary bed,wherein presence of detection reagent on the capillary bed indicatespresence of said at least one protein present in said biological sample.2. The method of claim 1, wherein said mammal is suspected of having aprotein-misfolding disorder or at risk of having a protein-misfoldingdisorder.
 3. The method of claim 2, wherein said at least one protein isselected from the group consisting of a misfolded protein, an aggregatedprotein, a supramolecular aggregated protein, a fragment of a misfoldedprotein, a fragment of an aggregated protein, a fragment of asupramolecular aggregated protein, mixtures thereof, and fragments ofsaid mixtures.
 4. The method of claim 3, wherein said misfolded proteinis selected from the group consisting of alpha-1 antitrypsin (SerpinA1),ceruloplasmin, heavy-chain IgG, light-chain IgG, interferon-inducibleprotein 6-16 (IF16-6,G1P3), albumin, and fragments thereof.
 5. Themethod of claim 2, wherein said protein-misfolding disorder is selectedfrom the group consisting of preeclampsia, Alzheimer's disease, priondisease and Parkinson's disease.
 6. The method of claim 5, wherein saidpreeclampsia is selected from the group consisting of mild preeclampsia,severe preeclampsia, atypical preeclampsia, hemodialysis-elevated liverenzyme-low platelet count (HELLP) syndrome and eclampsia.
 7. The methodof claim 2, wherein said biological sample is selected from the groupconsisting of urine, blood, saliva, tissue, interstitial fluid, serum,plasma, cerebrospinal fluid, amniotic fluid and an extracted substance.8. The method of claim 2, wherein said mammal is pregnant.
 9. The methodof claim 8, wherein said pregnant mammal is selected from the groupconsisting of a human, a primate and a genetically-engineered mammal.10. The method of claim 9, wherein said mammal is human.
 11. The methodof claim 10, wherein said human is approximately 8 to 42 weeks pregnant.12. The method of claim 2, wherein said mammal is postpartum.
 13. Themethod of claim 3, wherein said at least one protein may be detected byvisualization in order to obtain a qualitative result or asemi-quantitative result or detected by measurement in order to obtain asemi-quantitative or a quantitative result.